Plasmid shuttle vector between eschelichia coli and bacteria of genus brevibacillus

ABSTRACT

The invention provides a plasmid shuttle vector characterized by being replicable in both of bacteria of the genus Brevibacillus and  Escherichia coli , and by comprising a promoter capable of functioning in bacteria of the genus Brevibacillus and a DNA sequence(s) encoding a sequence(s) capable of functioning as a selective marker(s) for  Escherichia coli  and bacteria of the genus Brevibacillus, and a method of transforming bacteria of the genus Brevibacillus using the said plasmid shuttle vector.  
     The plasmid shuttle vector enables the efficient transformation of bacteria of the genus Brevibacillus. Further, since plasmid shuttle vector pNCMO2 of the invention in particular has a gene expression control region which efficiently expresses and secretes an objective gene in bacteria of the genus Brevibacillus but significantly suppresses the expression in  Escherichia coli , a recombinant gene expression plasmid can efficiently be constructed using  Escherichia coli , and it is suited for protein production in bacteria of the genus Brevibacillus.

TECHNICAL FIELD TO WHICH THE INVENTION BELONGS

[0001] The present invention relates to a plasmid shuttle vector betweenEscherichia coli and bacteria of the genus Brevibacillus and a method oftransforming bacteria of the genus Brevibacillus using the plasmidshuttle vector. More specifically, it relates to a plasmid shuttlevector which has a gene expression control region effectively expressingand secreting an objective gene in bacteria of the genus Brevibacillusbut significantly suppressing the expression in Escherichia coli, andwhich is suited for gene expression in bacteria of the genusBrevibacillus. Further, it relates to a process for producing a protein,characterized by culturing a transformant transformed by thetransformation method.

PRIOR ART

[0002] Udaka et al. have succeeded in the development of an expressionsystem of a recombinant protein using as a host bacteria of the genusBrevibacillus (separated from the ordinary classification of the genusBacillus) having excellent characteristics, not found in Escherichiacoli and Bacillus subtilis, that a large amount of a protein is secretedextracellularly and an extracellular protease activity is weak (PatentNo. 2082727, JP-A 62-201583 (1987), Yamagata, H. et al., J. Bacteriol.,169, 1239-1245 (1987), Shigezo Udaka, Nippon Nogeikagaku Kaishi 61,669-676 (1987), Takao, M. et al., Appl. Microbiol, Biotechnol., 30,75-80 (1989), Yamagata, H. et al., Proc. Natl. Acad. Sci., USA 86,3589-3593 (1989)). Production of α-amylase (Patent No. 2082727) or humanepidermal growth factor (hEGF) (Patent No. 2787585) by an expressionsystem using bacteria of the genus Brevibacillus as a host, and the likehave been so far reported.

[0003] Meanwhile, as a plasmid vector useful for gene expression ofbacteria of the genus Brevibacillus, for example, pNU200 (Shigezo Udaka,Nippon Nogeikagaku Kaishi 61, 669-676 (1987)), pNH300 (Shiga, Y. et al.,Applied and Environmental Microbiology, 58, 525-531 (1992)), pNH400(Ishihara, T, et al., J. Bacteriol., 177, 745-749 (1995)), pHY700 (JP-A4-278091 (1992), pHT series plasmids (Patent No. 2727391) and the likehave been so far reported. Especially, the present inventors appliedpNY301 and other pNY series plasmids for patent (JP-A 10-295378) as aplasmid vector useful for transformation of bacteria of the genusBrevibacillus.

PROBLEMS THAT THE INVENTION IS TO SOLVE

[0004] As mentioned above, the secretory production system of proteinsusing bacteria of the genus Brevibacillus as a host is one of usefulsystems in production of proteins by genetic recombination. However, atransformation efficiency of bacteria of the genus Brevibacillus is lowin comparison to a transformation efficiency of Escherichia coli.Accordingly, there was a defect that construction of recombinant geneexpression plasmids using bacteria of the genus Brevibacillus isdifficult in comparison to Escherichia coli. For example, in case ofusing an electroporation method (Takagi, T. et al, Agric. Biol. Chem.,53, 3099-3100 (1989)), a transformation efficiency of Escherichia colireaches 10¹⁰ CFU/ugDNA. Meanwhile, in case of Brevibacillus choshinensis(Bacillus brevis before; there was a change in taxonomical position inShida O., et al., Int. J. Syst. Bacteriol., 46, 939-946 (1996))belonging to the genus Brevibacillus, it was only 10⁷ CFU/ugDNA.Further, it has been known that in case of employing a method except theelectroporation method, the transformation efficiency of bacteria of thegenus Brevibacillus is more reduced.

[0005] Especially, transformation of bacteria of the genus Brevibacilluswith a gene encoding a protein having a complex higher-order structure,for example, a gene encoding a protein such as cholera toxin (CT)produced from Vibrio cholerae comprising one A subunit and five Bsubunits (Clements and Finkelstein, Intect. Immun., 24; 760-769 (1979))and protein production using the transformant have to date experienced agreat many difficulties.

[0006] As one of measures to cope with the problems, a method isconsidered in which a plasmid vector (hereinafter referred to as a“shuttle vector”) replicable also in other host cells easy of atransformation step, for example, Escherichia coli is used, constructionof a recombinant gene expression plasmid using the host cells is firstconstructed and transformation of bacteria of the genus Brevibacillus isfurther conducted by electroporation or the like using the recombinantgene expression plasmid.

[0007] A shuttle vector and a transformation method using the shuttlevector are techniques known to those skilled in the art. For example,with respect to a shuttle vector between Escherichia coli and Bacillussubtilis, JP-A 63-198986 (1988), JP-A 2000-197491 and the like have beenreported. Nevertheless, a plasmid shuttle vector utilizable in atransformation procedure by genetic recombination of bacteria of thegenus Brevibacillus and in recombinant gene expression with the transformant, and a method of transforming bacteria of the genusBrevibacillus using the said plasmid shuttle vector have been to dateunknown.

MEANS FOR SOLVING THE PROBLEMS

[0008] The present inventors have focussed on such usefulness that anobjective protein is extracellularly secreted in an expression systemusing bacteria of the genus Brevibacillus as a host, have seriouslyconsidered the necessity for the development of a novel host-vectorsystem that solves the foregoing defects, and have tried to develop anovel plasmid shuttle vector in which Escherichia coli and bacteria ofthe genus Brevibacillus can be used as a host and which can be used ingenetic recombination.

[0009] Thus, in order to attain the object, the present inventors havefocussed again on the point that especially plasmid vector pNY301 whichthe present inventors have already applied for patent, among a largenumber of plasmid vectors, contains a promoter and a secretion signalsequence from Brevibacillus choshinensis HPD31. Based on this plasmidvector pNY301, they have synthesized various DNA sequences by PCR or thelike, or cut them out from known sequences, further inserted these DNAfragments into the plasmid vector and confirmed the insertion. Moreover,they have performed confirmation of transformation with the resultingplasmids, confirmation of expression in transformants, and so forth. Asa result of performing a huge number of these delicate treatments andoperations with trial and error, they have finally succeeded inproduction of a desired novel plasmid shuttle vector.

[0010] The novel plasmid shuttle vector which has been successfullyproduced in this manner has been introduced into Escherichia coli andbacteria of the genus Brevibacillus, and the resulting bacteria havebeen cultured. Consequently, replication of the plasmid shuttle vectorhas been performed in any of the hosts. It has been found that in therecombinant gene expression using the plasmid shuttle vector, anobjective gene is effectively secreted and expressed in bacteria of thegenus Brevibacillus, but the expression is significantly suppressed inEscherichia coli. It has been further confirmed that even though atransformant of bacteria of the genus Brevibacillus is not obtained byother known methods, the transformation is enabled with the use of theplasmid shuttle vector. Thus, the invention has been completed.

[0011] That is, for solving the foregoing problems, the inventionprovides a plasmid shuttle vector between Escherichia coli and bacteriaof the genus Brevibacillus which is useful for transformation by geneticrecombination of bacteria of the genus Brevibacillus and for proteinproduction with the transformant, and a transformation method using theplasmid shuttle vector. Especially, it provides a plasmid shuttle vectorhaving a gene expression control region which efficiently expresses andsecretes an objective gene in bacteria of the genus Brevibacillus, butwhich significantly suppresses the expression in Escherichia coli, andbeing suited for gene expression in bacteria of the genus Brevibacillusand for protein production. Further, the invention provides a method oftransforming bacteria of the genus Brevibacillus using the plasmidshuttle vector, and a process for producing a protein, characterized byculturing bacteria of the genus Brevibacillus transformed with theplasmid shuttle vector.

BRIEF DESCRIPTION OF THE DRAWINGS

[0012]FIG. 1 shows a P2 promoter.

[0013]FIG. 2 shows a Lac operator sequence.

[0014]FIG. 3 shows an SD sequence (SD1).

[0015]FIG. 4 shows an SD sequence (SD2).

[0016]FIG. 5 shows an R2L6 type modified signal peptide (nucleotidesequence in upper column, and amino acid sequence in lower column).

[0017]FIG. 6 shows PCR cloning primer 1.

[0018]FIG. 7 shows PCR cloning primer 2.

[0019]FIG. 8 shows PCR cloning primer 3.

[0020]FIG. 9 shows PCR cloning primer 4.

[0021]FIG. 10 shows PCR cloning primer 5.

[0022]FIG. 11 shows PCR cloning primer 6.

[0023]FIG. 12 shows PCR cloning primer 7.

[0024]FIG. 13 shows PCR cloning primer 8.

[0025]FIG. 14 shows PCR cloning primer 9.

[0026]FIG. 15 shows PCR cloning primer 10.

[0027]FIG. 16 shows PCR cloning primer 11.

[0028]FIG. 17 shows PCR cloning primer 12.

[0029]FIG. 18 shows PCR cloning primer 13.

[0030]FIG. 19 shows PCR cloning primer 14.

[0031]FIG. 20 shows PCR cloning primer 15.

[0032]FIG. 21 shows a restriction map of plasmid shuttle vector pNCMO2.

[0033]FIG. 22 shows a process of construction of plasmid shuttle vectorpNCMO2.

[0034]FIG. 23 shows a continuation of the above.

[0035]FIG. 24 shows a process of construction of recombinant geneexpression plasmids pNCMO2BLA and pNC301BLA.

[0036]FIG. 25 shows a productivity of E. coli JM109/pNCMO2BLA and E.coli JM109/pNC301BLA.

[0037]FIG. 26 is a photo in place of a drawing, showing a haloformability and growth by E. coli JM109/pNCMO2BLA and E. coliMP109/pNC301BLA.

[0038]FIG. 27 is a photo in place of a drawing, showing effects of IPTGwith respect to BLA expression by E. coli JM109/pNCMO2BLA.

[0039]FIG. 28 shows a productivity of BLA by Brevibacillus choshinensisHPD31-S5/pNCMO2BLA and Brevibacillus choshinensis HPD31-S5/pNC301BLA.

[0040]FIG. 29 shows a process of construction of recombinant geneexpression plasmid pNCMO2CT.

[0041]FIG. 30 is photos in place of a drawing, showing a CoomassieBrilliant Blue stain image and a western blotting image of a culture oftransformant Brevibacillus choshinensis HPD31-S5/pNCMO2CT.

MODE FOR CARRYING OUT THE INVENTION

[0042] The invention is described in detail below.

[0043] The plasmid shuttle vector of the invention is a plasmid shuttlevector replicable in both of Escherichia coli and bacteria of the genusBrevibacillus, and comprising a promoter capable of functioning inbacteria of the genus Brevibacillus, and a DNA sequence(s) encoding aselective marker(s) for bacteria of the genus Brevibacillus andEscherichia coli.

[0044] The plasmid shuttle vector of the invention is autonomouslyreplicable in Escherichia coli and bacteria of the genus Brevibacillus.Accordingly, a recombinant gene expression plasmid can be constructed bya step such as insertion of a DNA fragment in the plasmid shuttle vectorof the invention using Escherichia coli, and further transformation ofbacteria of the genus Brevibacillus can be performed using therecombinant gene expression plasmid.

[0045] The promoter capable of functioning in bacteria of the genusBrevibacillus, which the plasmid shuttle vector of the inventionpossesses, is not particularly limited so long as it functions inbacteria of the genus Brevibacillus. It is preferably a promoter frombacteria of the genus Brevibacillus. Especially preferable examplesthereof can include promoters contained in an MWP promoter region fromBrevibacillus brevis 47 (Bacillus brevis 47 before)(JP-B 1-58950 (1989)and JP-B 7-108224 (1995)) and an HWP promoter region from Brevibacilluschoshinensis HPD31 (FERM BP-6863)(Bacillus brevis before; this strain isthe same as Bacillus brevis H102 (FERM BP-1087)(JP-A4-278091 (1992) andJP-A6-133782 (1994)), for example, a P2 promoter (Sequence No. 1: FIG.1).

[0046] The plasmid shuttle vector of the invention further comprises aribosome-binding region (SD sequence) and a DNA sequence encoding asignal peptide for secreting an expression protein at the 3′-terminus ofa promoter functioning in bacteria of the genus Brevibacillus. As the SDsequence, for example, a sequence from an HWP gene expression controlregion of Brevibacillus choshinensis, such as SD1 (Sequence No. 3: FIG.3) or SD2 (sequence No. 4: FIG. 4) can be used. As the secretion signalpeptide, for example, a signal peptide contained in an HWP promoterregion of Brevibacillus choshinensis HPD31 (FERM BP-1087, FERM BP-6863)can be used. As an especially preferable example, a modified HWP signalsequence of R2L6 type or the like (JP-A 7-170984 (1995)) can bementioned (with respect to this R2L6 type modified signal peptide, itsamino acid sequence is shown in Sequence No. 5, and the DNA sequenceencoding the same is shown in Sequence No. 21. Further, both of thesequences are shown in FIG. 5. In the drawing, the upper column shows anucleotide sequence, and the lower column shows an amino acid sequence).

[0047] The plasmid shuttle vector of the invention further comprises theDNA sequence encoding a replication control region necessary forreplication in bacteria. With respect to the replication control region,either one common region or different regions may be used for both ofbacteria of the genus Brevibacillus and Escherichia coli. Thereplication control region in bacteria of the genus Brevibacilluscomprises a Rep protein gene and an origin of replication. The sequencecapable of functioning as a replication control region in bacteria ofthe genus Brevibacillus is not particularly limited so long as it iscontained in a plasmid which is replicated and grown in bacteria of thegenus Brevibacillus and is a DNA sequence functioning as a replicationcontrol region of a plasmid. As an especially preferable example, a Repprotein gene from plasmid pUB110 and a DNA sequence encoding an originof replication can be mentioned.

[0048] In Escherichia coli, an origin of replication is required as areplication control region. A DNA sequence encoding an origin ofreplication capable of functioning in Escherichia coli is notparticularly limited so long as it is a DNA sequence contained in aplasmid replicated and grown in Escherichia coli and functioning as anorigin of replication of a plasmid. Preferable is a DNA sequencecontaining an OR1 region functioning as an origin of replication of aplasmid. Especially preferable is a DNA sequence encoding an ORI regionof plasmid vectors ordinarily used in a genetic recombinationmanipulation using Escherichia coli, such as pUC series, for example,pUC18, pUC118 and pUC119, and pBR322.

[0049] As a DNA sequence encoding a replication control region capableof functioning in both of bacteria of the genus Brevibacillus andEscherichia coli, a replication control region of a plasmid belonging toa pLS1/pE194 plasmid family, which is a replication control regioncapable of functioning in both of Gram-positive bacteria andGram-negative bacteria (Del Solar, G. et al., Mol. Microbiol., 8,789-796 (1993)) can be mentioned. For example, a DNA sequence encoding areplication control region (Sano. K. et al., FEMS Microbiology Letters,148, 223-226 (1997)) comprising ori, RepA and RepB of plasmid pLA106from Lactobacillus acidophilus can be used.

[0050] With respect to sequences capable of functioning as selectivemarkers for bacteria of the genus Brevibacillus and Escherichia coli,which the plasmid shuttle vector of the invention possesses, one commonsequence or different sequences may be used to both of bacteria of thegenus Brevibacillus and Escherichia coli. With respect to the sequencescapable of functioning as the selective markers, a drug resistance genecan be used. For bacteria of the genus Brevibacillus, for example, a DNAsequence containing a drug resistance gene from bacteria of the genusBrevibacillus and bacteria of the genus Bacillus analogous thereto canbe used. Especially preferable examples thereof can include a neomycinresistance gene contained in plasmid vector pNY301 (JP-A 10-295378(1998)) and an erythromycin resistance gene contained in pHT110 (PatentNo. 2727391). Further, for Escherichia coli, for example, a DNA sequenceencoding a kanamycin resistance gene, an ampicillin resistance gene, achloramphenicol resistance gene or the like which is a known drugresistance gene from Escherichia coli is available. Still further, as adrug resistance gene capable of functioning in both of bacteria of thegenus Brevibacillus and Escherichia coli, a zeocin resistance gene orthe like can be used.

[0051] The plasmid shuttle vector of the invention can further comprisea DNA sequence having a function to suppress expression of recombinantgene in Escherichia coli. For example, in case of Escherichia colihaving a lacI gene, a lac operator sequence (Sequence No. 2: FIG. 2)from Escherichia coli can be contained in the 3′-terminus of a promotersequence functioning in bacteria of the genus Brevibacillus.

[0052] The plasmid shuttle vector of the invention is novel. Forexample, pNCMO2 is a plasmid vector having a size of 5,224 bp. Arestriction map of pNCMO2 and a nucleotide sequence of its partcontaining a promoter were shown in FIG. 21. The promoter region ofpNCMO2 contains a P2 promoter from an HWP promotor region ofBrevibacillus choshinensis HPD31, a lac operator from Escherichia coli,an SD sequence, an R2L6 type modified signal peptide as a secretionsignal peptide, a multicloning site and an HS gene (JP-A 9-224677(1997)) as a terminator. With respect to the multicloning site, PstI,BamHI, SalI, XbaI, XhoI, EcoRI, KpnI, SmaI, ClaI, HindIII sites areavailable as cloning sites.

[0053] Plasmid shuttle vector pNCMO2 contains a DNA sequence encoding aneomycin resistance gene as a selective marker gene for bacteria of thegenus Brevibacillus, a Rep protein gene from pUB110 necessary forreplication in bacteria of the genus Brevibacillus, a ColE1 ori regionas an origin of replication of Escherichia coli and an ampicillinresistance gene as a selective marker gene for Escherichia coli.

[0054] In the construction of plasmid shuttle vector pNCMO2 in theinvention, pNY301, pNH326, pNU201 and the like are used. Plasmid pNY301is laid open in JP-A 10-295378 (1998), plasmid pNH326 in Kajino., T., etal., Appl. Environ. Microbiol., 66, 638-642 (2000), and pNU201 in Udaka,S. et al., Methods in Enzymology, 217, 23-33 (1993) respectively, andthese are known plasmids. Other plasmids which are used in constructionof the plasmid shuttle vector of the invention can be procured on themarket.

[0055]Brevibacillus choshinensis HPD31-S5/pNCMO2 carrying plasmid vectorpNCMO2 was internationally deposited on Dec. 12, 2000 in PatentMicroorganism Depository of National Institute of Bioscience andHuman-Technology, Agency of Industrial Science and Technology, Ministryof International Trade and Industry (the present name: InternationalPatent Organism Depositary, National Institute of Advanced IndustrialScience and Technology), and it is assigned Receipt No. FERM BP-7394.

[0056] The host used in construction of the expression plasmid of theinvention is not particularly limited so long as it is a strainbelonging to Escherichia coli. As an especially preferable example, E.coli JM109 can be mentioned. E. coli JM109 is a known strain which canbe procured on the market.

[0057] With respect to a method of constructing a recombinant geneexpression plasmid from the plasmid shuttle vector of the inventionusing Escherichia coli, a method based on a standard technology ofmolecular biology known to a skilled person can be used properly. Forexample, the method described in Molecular Cloning 2nd ed., A LaboratoryManual, Cold Spring Harbor Laboratory (1989)) is mentioned. The detailsare described in Examples.

[0058] As the host used in the expression of the recombinant gene in theinvention, any strain of bacteria of the genus Brevibacillus can beused. Preferable is Brevibacillus choshinensis. Especially preferableexamples can include Brevibacillus choshinensis HPD31 (FERM BP-1087,FERM BP-6863), and Brevibacillus choshinensis HPD31-S5 (FERM BP-6623)which is a variant thereof.

[0059] As a method of transforming bacteria of the genus Brevibacillususing the shuttle vector of the invention and a recombinant geneexpression plasmid constructed with Escherichia coli, a knowntransformation method known to a skilled person, such aselectroporation, can be used.

[0060] A medium used in culture of the transformant of the inventioncontains, as required, a carbon source, a nitrogen source and inorganicsalts. Culture may be conducted using a synthetic medium made mainly ofsaccharide and inorganic salts. In case of using a strain exhibitingauxotrophy, it is advisable to add nutrients required for its growth toa medium. Further, antibiotic, antifoam and the like may be added asrequired.

[0061] With respect to culture conditions, the initial pH of a medium iscontrolled to from 5.0 to 9.0, preferably from 6.5 to 7.5. The culturetemperature is usually from 15° C. to 42° C., preferably from 24 to 37°C., and the culture time is usually from 16 to 360 hours, preferablyfrom 24 to 144 hours. Bacteria of the genus Brevibacillus transformed bythe method of the invention thereby produce and accumulate proteins in aculture solution.

[0062] After completion of the culture, collection of an objectiveprotein from the culture is enabled by an appropriate combination ofprotein purification methods known to a skilled person, such as solventextraction, ultrafiltration, ammonium sulfate fractionation, HPLC, gelfiltration chromatography, ion exchange chromatography, affinitychromatography, hydrophobic interaction chromatography, electrophoresisand isoelectric focussing.

EXAMPLES

[0063] The invention is illustrated more specifically below by referringto Examples. However, this is illustrative, and the invention is notlimited thereto.

Example 1

[0064] Construction of Plasmid Shuttle Vector pNCMO2

[0065] (1) Construction of Plasmid Shuttle Vector pNC301

[0066] Amplification was conducted by PCR with plasmid vector pUC119 asa template using primer 1 (Sequence No. 6: FIG. 6) and primer 2(Sequence No. 7: FIG. 7), and the resulting PCR product was cleaved withNsiI to obtain a DNA fragment of approximately 2 kbp containing a ColE1ori region as an origin of replication of Escherichia coli and anampicillin resistance gene.

[0067] PCR was conducted such that a PCR kit (made by Takara Shuzo Co.,Ltd.) was used, 100 pmol of each primer, 2.5 units of a Taq polymerase,200 μM dNTP, 1 ng pUC119 template DNA and 100 μl Taq buffer (10 mMTris-hydrochloride (pH 8.5), 2.5 mM Mg⁺⁺, 50 mM potassium chloride and100 μg/ml bovine serum albumin) were mixed, the mixture was maintainedat 96° C. for 30 seconds, and a cycle of DNA thermal denaturation (94°C., 60 sec), primer annealing (54° C., 60 sec) and primer elongation(70° C., 60 sec) was then repeated 25 times. This condition ishereinafter referred to as condition 1.

[0068] The above-obtained DNA fragment of approximately 2 kbp wasligated to an Sse8387I site of plasmid vector pNY301 (JP-A 10-295376(1998)) containing a P5 promoter from an HWP promoter region ofBrevibacillus choshinensis HPD31 and a natural type HWP signal peptidesequence as a secretion signal peptide with a T4 DNA ligase using a DNAligation kit (made by Takara Shuzo Co., Ltd.). E. coli JM109 (made byTakara Shuzo Co., Ltd.) was transformed with this DNA by the CaCl₂method (Molecular Cloning 2nd ed., A Laboratory Manual, Cold SpringHarbor Laboratory, 1, 82, (1989)), and a plasmid was extracted from theresulting ampicillin resistance transformant. The resulting plasmid wasa novel plasmid shuttle vector replicable in Escherichia coli andbacteria of the genus Brevibacillus, and was designated pNC301.

[0069] Plasmid shuttle vector pNC301 contains the P5 promoter from theHWP promoter region of Brevibacillus choshinensis HPD31, the neomycinresistance gene as a selective marker gene for the genus Brevibacillus,the ColE1 ori region as an origin of replication of Escherichia coli andthe ampicillin resistance gene as a selective marker gene forEscherichia coli. It contains further the natural type HWP signalpeptide as a secretion signal peptide (FIG. 22).

[0070] Construction of plasmid shuttle vector pNCMO2 is described belowby referring to FIG. 23.

[0071] (2) Construction of Plasmid pNC

[0072] The total length of pNC301 obtained by excluding from plasmidshuttle vector pNC301 the HWP promoter region from Brevibacilluschoshinensis HPD31 was amplified by PCR under condition 1 using primer 3(Sequence No. 8: FIG. 8) and primer 4 (Sequence No. 9: FIG. 9). Theamplified fragment of approximately 5 kbp was treated with BclI, andsubjected to self-ligation with a T4 DNA ligase. E. coli JM109 wastransformed with this DNA by the CaCl₂ method to obtain plasmid pNC. Thereaction condition of PCR was (a cycle of a denaturation temperature:94° C.-60 sec, an annealing temperature: 54° C.-120 sec and a DNA chainelongation temperature: 70° C.-180 sec was repeated 25 times).

[0073] (3) Construction of Plasmid pGEM-TP2

[0074] A DNA sequence encoding a P2 promoter from an HWP promoter regioncontained in plasmid pNU210 (Udaka, S. et al., Methods In Enzymology,217, 23-33 (1993)) was amplified by PCR under condition 1 using primer 5(Sequence No. 10: FIG. 10) and primer 6 (Sequence No. 11: FIG. 11) toobtain a DNA fragment of approximately 140 bp. Since primer 6 contains alac operator sequence, this DNA fragment contains the lac operatorsequence at the 3′-terminus of the P2 promoter. This fragment wasligated to pGEM-T (made by Promega) with a T4 DNA ligase. E. coli JM109was transformed with this DNA by the CaCl₂ method, and a plasmid wasextracted from the resulting ampicillin resistance transformant toobtain pGEM-TP2.

[0075] (4) Construction of Plasmid pGEM-TP2⁺

[0076] A DNA sequence contained in plasmid pNH326 (Kajino, T., et al.,Appl. Environ, Microbiol., 66, 638-642 (2000)), which contains SD1 andSD2 sequences of an HWP promoter region of Brevibacillus choshinensisHPD31, an R2L6 type modified HWP signal sequence (JP-A 7-170984(1995))(Sequence No. 5, Sequence No. 21: FIG. 5) as a secretion signalpeptide sequence and a multicloning site, was amplified by PCR undercondition 1 using primer 7 (Sequence No. 12: FIG. 12) and primer 8(Sequence No. 13: FIG. 13) to obtain a DNA fragment of approximately 270bp. The resulting DNA fragment was treated with restrictionendonucleases NsiI and HindIII, and ligated to a PstI/HindIII site ofpGEM-TP2 with a T4DNA ligase. E. coli JM109 was transformed with thisDNA by the competent cell method to obtain pGEM-TP2⁺.

[0077] (5) Construction of Plasmid Shuttle Vector pNCMO2

[0078] Further, pGEM-TP2+was treated with BamHI and PstI. The resultingfragment of approximately 400 bp was inserted into a BclI/PstI site ofpNC, ligated with a T4 DNA ligase, and E. coli JM109 was transformedwith this DNA by the CaCl₂ method. A plasmid was extracted from theresulting ampicillin resistant strain to obtain novel plasmid shuttlevector pNCMO2 (FIG. 23). The construction outline of plasmid shuttlevector pNCMO2 is shown in FIGS. 22 and 23. The restriction map of thethus-obtained novel plasmid shuttle vector pNCMO2 and the nucleotidesequence of its part containing a promoter and the like were shown inFIG. 21. In the above-mentioned manner, Brevibacillus choshinensisHPD31-S5 (FERM BP-6623) was transformed using this plasmid shuttlevector, and the resulting transformant (Brevibacillus choshinensisHPD31S5/pNCMO2) was internationally deposited as FERM BP-7394.

Example 2

[0079] Transformation Efficiency of pNCMO2 and pNC301

[0080] A transformation efficiency of pNCMO2 and pNC301 is shown inTable 1. The transformation efficiency of both vectors to E. coli JM109was satisfactorily high. The transformation efficiency of pNCMO2 toBrevibacillus choshinensis HPD31-S5 was low, as compared with that ofpNC301, but high enough to insert the constructed plasmid. Thetransformation of E. coli JM109 was performed by the CaCl₂ method(Molecular Cloning, Cold Spring Harbor Laboratory, Press, 1, 82,(1989)), and the transformation of Brevibacillus choshinensis HPD31-S5was performed by electroporation (Takagi, H. et al., Agric. Biol. Chem.,53, 3099-3100 (1989)). The electroporation was performed underconditions of 1.5 kV, 1000 Ω, 25 μF and 1.8 msec using Gene Pulser(manufactured by BioRad). TABLE 1 Transformation efficiency of pNCMO2and pNC301 Transformation efficiency Plasmid vector Host bacterium(CFU/μgDNA) pNOMO2 E. coli JM109 6.1 × 10⁶ pNC301 E. coli JM109 8.6 ×10⁶ pUC119 E. coli JM109 1.9 × 10⁷ pNCMO2 Brevibacillus choshinensisHPD31-S5 5.2 × 10⁴ pNC301 Brevibacillus choshinensis HPD31-S5 1.2 × 10⁶pNY301 Brevibacillus choshinensis HPD31-S5 1.9 × 10⁶

Example 3

[0081] Comparison of BLA Production Between Plasmid Shuttle VectorpNCMO2 and Plasmid Shuttle Vector pNC301

[0082] (1) Construction of Recombinant Gene Expression PlasmidspNCMO2BLA and pNC301BLA

[0083] An α-amylase (BLA) gene from Bacillus licheniformis was amplifiedby PCR with pHY4631 (Yamagata, H. et al., J. of Biotechnol, 169,1239-1245 (1987)) as a template using primer 9 (Sequence No. 14: FIG.14) and primer 10 (Sequence No. 15: FIG. 15). The resulting PCR productwas digested with restriction endonucleases PstI and HindIII to obtain aBLA gene-containing DNA fragment of approximately 1.5 kb. Plasmidvectors pNCMO2 and pNC301 constructed in Example 1 were digested withrestriction endonucleases PstI and HindIII, and the above-obtained BLAgene-containing fragment was ligated thereto with a T4DNA ligase toobtain plasmid vectors pNCMO2BLA and pNC301BLA (FIG. 24). Further, E.coli JM109 was transformed by the CaCl₂ method using plasmid vectorspNCMO2BLA and pNC301BLA to obtain transformants E. coli JM109/pNCMO2BLAand E. coli JM109/pNC301BLA carrying plasmid vectors pNCMO2BLA andpNC301BLA respectively.

[0084] Further, as a BLA gene, it was also possible to use a DNAfragment cleaved from a BLA gene-containing plasmid, for example,pHT110BLA (Patent No. 2727391).

[0085] (2) BLA Production with E. coli JM109/pNCMO2BLA and E. coliJM109/pNC301BLA

[0086] Each of the transformants was inoculated to 1.5 ml portion of2×YT medium, an incubated overnight at 37° C. with shaking. This culturesolution was treated with a sonicator for 30 seconds to disrupt thecells, and the amylase activity in the treated solution was measured bythe Saito's-method (Arch. Biochem. Biophys., 155, 290 (1973)) using asoluble starch as a substrate (FIG. 25). E. coli JM109/pNCMO2BLAproduced amylase in an amount which was approximately 1/70 in comparisonto E. coli JM109/pNC301BLA, and the gene expression in Escherichia coliwas efficiently suppressed.

[0087] Further, E. coli JM109/pNCMO2BLA and E. coli JM109/pNC301BLAwereinoculated on an LAagar medium containing 3% starch, and incubatedovernight at 37° C. to form colonies, and a halo formability and growththereof were compared (FIG. 26: photo in place of a drawing). As aresult, E. coli JM109/pNCMO2BLA was smaller in size of the halo aroundthe colony than E. coli JM109/pNC301BLA, and the BLA expression wastherefore suppressed. Moreover, from the size of the colony grown, itwas found that E. coli JM109/pNCMO2BLA was proliferated faster. This ispresumably because the BLA expression was suppressed at good efficiencyin E. coli JM109/pNCMO2BLA and the stress exerted on the strain wastherefore reduced to improve the amplification.

[0088] When E. coli JM109/pNCMO2BLA was incubated on an agar mediumcontaining 1 mM IPTG, the size of the halo formed was almost the same asthat of the halo formed with the same strain incubated on a IPTG-freeagar medium (FIG. 27: photo in place of a drawing). With respect to theexpression suppressing effect in Escherichia coli provided by pNCMO2,this is presumably because in addition to the suppressing effect byinserting the lac operator, the P2 promoter activity is quite low inEscherichia coli, as compared with the P5 promoter activity.

[0089] (3) BLA Production with Brevibacillus choshinensisHPD31-S5/pNCMO2BLA and Brevibacillus choshinensis HPD31S5/pNC301BLA

[0090]Brevibacillus choshinensis HPD31-S5 was transformed byelectroporation with plasmid vectors pNCMO2BLA and pNC301BLA obtained in(1) respectively to obtain transformants Brevibacillus choshinensisHPD31-S5/pNCMO2BLA and Brevibacillus choshinensis HPD31-S5/pNC301BLAcarrying plasmid vectors pNCMO2BLA and pNC301BLA respectively (FIG. 24).The electroporation was performed under conditions of 1.5 kV, 1,000 Ω,25 μF and 1.8 msec using Gene Pulser (manufactured by BioRad). Each ofthe transformants was inoculated in a test tube containing 1.5 ml TMNmedium, and incubated at 30° C. for 2 days with shaking. This culturesolution was centrifuged, and the amylase activity of the culturesupernatant was measured by the Saito's method (Arch. Biochem. Biophys.,155, 290 (1973)) using a soluble starch as a substrate (FIG. 28).Brevibacillus choshinensis HPD31-S5/pNCMO2BLA produced amylase in anamount which was approximately 22 times as large as Brevibacilluschoshinensis HPD31-S5/pNC301BLA. Consequently, it was shown that pNCMO2can express a gene in bacteria of the genus Brevibacillus far moreefficiently than pNC301.

Example 4

[0091] Construction of Recombinant Gene Expression Plasmid pNY326CT

[0092] (1) Obtainment of CTA Gene and CTB Gene

[0093] A cholera toxin (CT) A subunit gene (CTA)(0.7 bp) was amplifiedby PCR with a Vibrio cholerae chromosomal DNA (Mekalanos, J. et al.,Nature, 306, 551-557 (1983)) as a template using two synthetic primers,primer 11 (Sequence No. 16: FIG. 16) and primer 12 (Sequence No. 17:FIG. 17). In like manner, a CT B subunit gene (CTB)(0.3 bp) wasamplified using two primers, primer 13 (Sequence No. 18: FIG. 18) andprimer 14 (Sequence No. 19: FIG. 19).

[0094] (2) Plasmid pT7BlueCTA and plasmid pT7BlueCTB

[0095] Plasmid pT7Blue (made by Novagen) and the above-obtained CTA geneor CTB gene were ligated with a T4 ligase to obtain plasmid pT7BlueCTAcarrying the CTA gene or plasmid pT7BlueCTB carrying the CTB gene.

[0096] (3) Construction of Plasmid pNY326CTA

[0097] Plasmid pNY326 [plasmid obtained by converting the signal peptideof pNY301 (JP-A 10-295378 (1998)) to an R2L6 type (Sequence Nos. 5, 21:FIG. 5)] was treated with NcoI and HindIII to obtain a fragment of 3.6kbp. Further, pT7BlueCTA was treated with NcoI and HindIII to obtain aCTA gene-containing DNA fragment of 0.7 kbp. This DNA fragment wasligated to the above-obtained gene fragment of 3.6 kbp with a T4 DNAligase. Brevibacillus choshinensis HPD31-S5 was transformed byelectroporation using the ligated DNA. From the resulting neomycinresistant strain, a plasmid was extracted to obtain pNY326CTA carryingthe CTA gene.

[0098] (4) Construction of Plasmid pNY326CTB

[0099] In like manner, pNY326 was treated with NcoI and BamHI to obtaina DNA fragment of 3.6 kbp. Further, pT7BlueCTB was treated with NcoI andBamHI to obtain a CTB gene-containing DNA fragment of 0.3 kbp. This DNAfragment was ligated to the above-obtained DNA fragment of 3.6 kbp witha T4 DNA ligase. Brevibacillus choshinensis HPD31-S5 was transformedusing the ligated DNA to select a strain having a neomycin resistance.From the selected strain, a plasmid was extracted to obtain plasmidpNY326 CTB carrying the CTB gene.

[0100] (5) Trial of Transformation of Brevibacillus choshinensisHPD31-S5 with pNY326CT

[0101] pNY326CTB was treated with BamHI and HindIII to obtain a DNAfragment of 4.7 kbp. Further, a CTA gene in the form containing a SD2sequence (SD-CTA gene (0.8 kbp)) was amplified by PCR with pNY326CTA asa template using two primers, primer 15 (Sequence No. 20: FIG. 20) andprimer 11 (Sequence No. 16: FIG. 16). The PCR product was treated withBamHI and HindIII to obtain a fragment of 0.4 kbp. This fragment wasligated to the above-obtained DNA fragment of 4.7 kbp from pNY326CTBwith a T4 DNA ligase. Brevibacillus choshinensis HPD31-S5 wastransformed by electroporation using the ligated DNA, and thetransformant was spread on a 50 μg/ml neomycin-containing TM agar medium(1% peptone, 0.2% yeast extract, 0.5% meat extract, 1% glucose, 0.001%FeSO₄.7H₂O, 0.001% MnSO₄.4H₂O, 0.0001% ZnSO₄.7H₂O, 1.5% agar, pH 7.0) totry to select a strain having a neomycin resistance. However, theresistant strain was not obtained. The electroporation was performedunder conditions of 1.5 kV, 1,000 Ω, 25 μF and 1.8 msec using GenePulser (manufactured by BioRad).

Example 5

[0102] Construction of Recombinant Gene Expression Plasmid pNCMO2CT

[0103] (1) Construction of Plasmid pNCMO2CTB.

[0104] pNCMO2 was treated with NcoI and BamHI to obtain a fragment of5.2 kbp. Further, pT7BlueCTB was treated with NcoI and BamHI to obtain aCTB gene-containing DNA fragment of 0.3 kbp. This DNA fragment wasligated to the above-obtained gene fragment of 5.2 kbp with a T4 DNAligase (FIG. 29). E. coli JM109 was transformed with this ligated DNA,and a plasmid was extracted to obtain pNCMO2CTB carrying the CTB gene.

[0105] (2) Construction of Plasmid pNCMO2CT

[0106] pNCMO2 CTB was treated with BamHI and HindIII to obtain afragment of 5.5 kbp. The SD-CTA gene (0.8 kbp) obtained in Example 4 (5)was treated with BamHI and HindIII to recover a fragment of 0.8 kbp.This fragment was ligated to the above-obtained gene fragment of 5.5 kbpwith a T4 DNA ligase (FIG. 29). E. coli JM109 was transformed with theligated DNA to select a strain having an ampicillin resistance. From theselected strain, a plasmid was extracted to obtain three strains ofplasmid pNCMO2CT carrying the CTB gene. As a result of sequencing, itwas found that in all of the strains, the CTB gene was correctly ligatedto the plasmid. The strains obtained here were designated E. coliJM109/pNCMO2CT. Brevibacillus choshinensis HPD31-S5 was transformed withpNCMO2CT extracted from E. coli JM109/pNCMO2CT by electroporation.Consequently, a large number of transformants carrying the same plasmidwere obtained. The resulting strains were designated Brevibacilluschoshinensis HPD31-S5/pNCMO2CT. The electroporation was performed underconditions of 1.5 kV, 1,000 Ω, 25 μF and 1.8 msec using Gene Pulser(manufactured by BioRad).

[0107] (3) Expression of CT with Brevibacillus choshinensisHPD31-S5/pNCMO2CT

[0108]Brevibacillus choshinensis HPD31-S5/pNCMO2CT was cultured in 3 mlof a 50 μg/ml neomycin-containing TM liquid medium at 30° C. for 2 daysusing a medium-sized test tube while being shaken, and the culturesupernatant was subjected to SDS-PAGE. The analysis was conducted byCoomassie Brilliant Blue staining and western blotting with a rabbit CTpolyclonal antibody. The estimated molecular weights of CTA and CTB were28 kDa and 12 kDa respectively, and showed staining bands in positionsof corresponding molecular weights respectively. From the color depthsof the bands, the secretory production amount of CTA was estimated at 70mg/l and the secretory production amount of CTB at 30 mg/l respectively.Its SDS-PAGE electrophoresis pattern is shown in a photo in place of adrawing (FIG. 30). Further, when the culture supernatant was purifiedusing a galactose resin specifically binding CT (Microbial Pathogenesis,16, 71-79 (1994)) and the purified fraction was then subjected toelectrophoresis without being heat-treated, the band was shifted to asite of approximately 90 kDa. From this fact, it was presumed that theexpressed subunit took a 1A5B structure.

EFFECTS OF THE INVENTION

[0109] According to the invention, the plasmid shuttle vector betweenEscherichia coli and bacteria of the genus Brevibacillus is provided.Further, the plasmid shuttle vector of the invention, as shown in theresults of the expression of the BLA gene in Example 3, suppresses thegene expression in Escherichia coli and can allow the efficient geneexpression in Brevibacillus choshinensis. As shown by the expression ofCT in Examples 4 and 5, plasmid shuttle vector pNCMO2 in the inventionis useful especially for construction of the recombinant gene expressionplasmid requiring complex gene construction and for protein productionwith the transformant transformed with the said recombinant geneexpression plasmid.

[0110] Description on Microorganisms Deposited Under Rule 13-2

[0111] 1. Brevibacillus choshinensis HPD31-S5/pNCMO2

[0112] a. Name and address of a depositary agency in which thismicroorganism was deposited

[0113] Name: International Patent Organism Depositary, NationalInstitute of Advanced Industrial Science and Technology

[0114] Address: AIST Tsukuba Central 6, 1-1-1 Higashi, Tsukuba Ibaraki,305-8566, Japan

[0115] b. Date on which the microorganism was deposited in thedepositary agency of a Dec. 12, 2000

[0116] c. Receipt Number that the depositary agency of a assigned on thedeposition FERM BP-7394

[0117] 2. Brevibacillus choshinensis HPD31 (FERM BP-1087)

[0118] a. Name and address of a depositary agency in which thismicroorganism was deposited

[0119] Name: International Patent Organism Depositary, NationalInstitute of Advanced Industrial Science and Technology

[0120] Address: AIST Tsukuba Central 6, 1-1-1 Higashi, Tsukuba Ibaraki,305-8566, Japan

[0121] b. Date on which the microorganism was deposited in thedepositary agency of a Aug. 31, 1999

[0122] c. Receipt Number that the depositary agency of a assigned on thedeposition FERM BP-6863

1 21 1 44 DNA Brevibacillus choshinensis 1 aaggcgccgc aacttttgattcgctcaggc gtttaatagg atgt 44 2 21 DNA Escherichia coli 2 aattgtgagcggataacaat t 21 3 10 DNA Brevibacillus choshinensis 3 gaaaggaggt 10 4 12DNA Brevibacillus choshinensis 4 agaggaggag aa 12 5 30 PRT ARTIFICIALSEQUENCE SYNTHETIC DNA 5 Met Lys Lys Arg Arg Val Val Asn Ser Val Leu LeuLeu Leu Leu Leu 1 5 10 15 Ala Ser Ala Leu Ala Leu Thr Val Ala Pro MetAla Phe Ala 20 25 30 6 22 DNA ARTIFICIAL SEQUENCE SYNTHETIC DNA 6aaaatgcatg gccagcaaaa gg 22 7 20 DNA ARTIFICIAL SEQUENCE SYNTHETIC DNA 7aaaatgcatg acgaaagggc 20 8 32 DNA ARTIFICIAL SEQUENCE SYNTHETIC DNA 8aaatgatcaa agcttcggca ttatagtgcg gg 32 9 30 DNA ARTIFICIAL SEQUENCESYNTHETIC DNA 9 aaatgatcct gcaggatccg tcgactctag 30 10 24 DNA ARTIFICIALSEQUENCE SYNTHETIC DNA 10 aaaggatccg acataatgga cagg 24 11 52 DNAARTIFICIAL SEQUENCE SYNTHETIC DNA 11 aaactgcaga ataattgtta tccgctcacaattacatcct attaaacgcc tg 52 12 26 DNA ARTIFICIAL SEQUENCE SYNTHETIC DNA12 aaactgcatg gctttcctgc gaaagg 26 13 23 DNA ARTIFICIAL SEQUENCESYNTHETIC DNA 13 aaaagcttat cgatttcgaa ggg 23 14 23 DNA ARTIFICIALSEQUENCE SYNTHETIC DNA 14 cgctgcagca gcggcggcaa atc 23 15 26 DNAARTIFICIAL SEQUENCE SYNTHETIC DNA 15 aaaagcttat ctttgaacat aaattg 26 1634 DNA ARTIFICIAL SEQUENCE SYNTHETIC DNA 16 aaccatggct ttcgctacagatgataagtt atat 34 17 28 DNA ARTIFICIAL SEQUENCE SYNTHETIC DNA 17ttaagcttca taattcatcc ttaattct 28 18 58 DNA ARTIFICIAL SEQUENCESYNTHETIC DNA 18 aaccatggct ttcgctacac ctcaaaatat tactgatttg tgtgcagaataccacaac 58 19 32 DNA ARTIFICIAL SEQUENCE SYNTHETIC DNA 19 aaggatccttaatttgccat actaattgcg gc 32 20 29 DNA ARTIFICIAL SEQUENCE SYNTHETIC DNA20 gcggatccag aggaggagaa cacaaggtc 29 21 90 DNA ARTIFICIAL SEQUENCESYNTHETIC DNA 21 atgaaaaaaa gaagggtcgt taacagtgta ttgcttctgc tactgctagctagtgcactc 60 gcacttactg ttgctcccat ggctttcgct 90

1. A plasmid shuttle vector characterized by being replicable in both ofbacteria of the genus Brevibacillus and Escherichia coli, and bycomprising a promoter capable of functioning in bacteria of the genusBrevibacillus, and a DNA sequence(s) encoding a sequence(s) capable offunctioning as a selective marker(s) for Escherichia coli and bacteriaof the genus Brevibacillus.
 2. A plasmid shuttle vector replicable inboth of Escherichia coli and bacteria of the genus Brevibacillus,characterized by comprising (a) a promoter capable of functioning inbacteria of the genus Brevibacillus, an SD sequence and a signalsequence, (b) a Rep protein gene and a DNA sequence encoding an originof replication which are capable of functioning in bacteria of the genusBrevibacillus, (c) a DNA sequence encoding a sequence capable offunctioning as a selective marker for bacteria of the genusBrevibacillus, (d) a DNA sequence encoding an origin of replicationcapable of functioning in Escherichia coli, and (e) a DNA sequenceencoding a sequence capable of functioning as a selective marker forEscherichia coli.
 3. The plasmid shuttle vector according to claim 1 or2, characterized in that the promoter capable of functioning in bacteriaof the genus Brevibacillus as defined in claim 1 or 2 is a P2 promoterthat an HWP promoter from Brevibacillus choshinensis includes, and itsnucleotide sequence is represented by Sequence No.
 1. 4. The plasmidshuttle vector according to any one of claims 1 to 3, characterized byfurther comprising a DNA sequence having a function of suppressingexpression of a recombinant protein in Escherichia coli.
 5. The plasmidshuttle vector according to claim 4, characterized in that the DNAsequence having the function of suppressing expression of a recombinantprotein in Escherichia coli as defined in claim 4 is a lac operatorsequence from Escherichia coli, and its nucleotide sequence isrepresented by Sequence No.
 2. 6. Plasmid shuttle vector pNCMO2 havingrestriction endonuclease regconition sites shown in FIG.
 21. 7.Brevibacillus choshinensis HPD31-S5/pNCMO2 strain containing plasmidshuttle vector pNCMO2 of claim 6 and deposited under InternationalDeposit No. FERM BP-7394.
 8. Brevibacillus choshinensis transformed witha recombinant plasmid obtained by inserting a DNA sequence of a geneencoding a protein into the plasmid shuttle vector as defined in any oneof claims 1 to
 6. 9. Brevibacillus choshinensis according to claim 8,characterized in that Brevibacillus choshinensis is Brevibacilluschoshinensis HPD31-S5 deposited under International Deposit No. FERMBP-6623.
 10. A transformation method characterized by transformingEscherichia coli with a recombinant plasmid obtained by inserting a DNAsequence of a gene encoding a protein into the plasmid shuttle vector asdefined in any one of claims 1 to 6, extracting a plasmid from theresulting transformant, and transforming bacteria of the genusBrevibacillus using the extracted plasmid.
 11. A process for producing aprotein, characterized by culturing bacteria of the genus Brevibacillustransformed with a recombinant plasmid obtained by inserting a DNAsequence of a gene encoding a protein into the plasmid shuttle vector asdefined in any one of claims 1 to 6, and collecting said objectiveprotein secreted and accumulated in the culture solution.